The invention relates to the use of apotransferrin that has bound divalent zinc or copper ions as an agent for prophylactic and therapeutic treatment of the toxic effects of endotoxins. Apotransferrin is a glycoprotein with an average molecular weight of 80,000 Dalton. It can bind mainly trivalent iron ions reversibly. It can also bind other metal ions instead of iron such as Cu(II), Mn(III), Co(III) or Zn(II). In human and animal organisms, apotransferrin usually occurs as transferrin, a protein-metal complex with trivalent iron.
The main function of apotransferrin is considered to be its ability to bind and transport specific trivalent iron ions, whereby it can bind and transport one or two molecules of iron reversibly. In this way, the iron is transported following its absorption in the small intestine to the iron depots in the liver and spleen as well as in the reticulocytes in the hematopoietic tissue. The normal range of plasma apotransferrin-iron complex (transferrin) concentration is 200 mg/dl to 400 mg/dl.
A further important function of transferrin in its iron-free form is considered to be its bacteriostatic effect (Martin CM, Jandl JH, Finland M. J Infect Dis 1963; 112:158-163; Fletcher J. Immunology 1971; 20:493-500). Iron is an essential growth factor for bacteria. The complexing of iron by transferrin keeps the free iron concentration in the plasma under the minimum required for bacterial growth. Berger und Beger (Berger D, Beger HG. Clin Chim Acta 1987; 163:289-299; Arzneim-Forsch/Drug Res 1988; 38:817-820; and Prog Clin Biol Res 1988; 272:115-124) concluded on the basis of the bacteriostatic effect of transferrin that transferrin in its iron-free form might even fulfil the function of an adjunctive anti-microbial agent in extensive gram-negative infections in the sense of a complement to conventional antibiotic therapy.
Apotransferrin that has bound trivalent iron, i.e. in its form as transferrin, is also capable of reducing the biological activity of endotoxins. The ability of transferrin to neutralize endotoxin increases along with the iron load as is described in detail in DE 38 42 143 and DE 38 44 667. The fact that apotransferrin, having bound iron ions, is more or less capable of neutralizing endotoxins, depending on the iron level, does not necessarily mean that the same effect will achieved when other metal ions are bound.
In contrast to the conclusions described in DE 38 42 143 and in DE 3844 667, Berger et al. report that ". . . the endotoxin binding capacity is restricted to apotransferrin." (Berger D, Winter M, Beger HG. Clin Chim Acta 1990; 189:1 (Summary)) and that the ability of apotransferrin to bind and neutralize endotoxin is reduced when iron is bound (Berger D, Beger HG. Clin Chim Acta 1987; 163:289-299; Arzneim-Forsch/Drug Res 1988; 38:817-820; and Prog Clin Biol Res 1988; 272: 115-124; Langenbecks Archiv fur Chirurgie 1990; Suppl. 1-6; Berger D, Winter M, Beger HG. Clin Chim Acta 1990, 189: p. 1-6). The authors even mention that "transferrin . . . exhibits endotoxicity enhancing activity." (Berger D, Winter M, Beger HG. Clin Chim Acta 1990; 189: p. 4). Berger et al. also report that, besides pH, the presence of divalent cations (Ca.sup.2+, Mg.sup.2+, Mn.sup.2+) in the reaction medium is of decisive importance regarding the binding of endotoxin by apotransferrin (Berger D, Beger HG. Arzneim-Forsch/Drug Res 1988; 38:817-820; Prog Clin Biol Res 1988; 272: 115-124; Berger D, Winter M, Beger HG. Clin Chim Acta 1990, 189: p. 1-6 and Berger D, Kitterer WR, Beger HG. Eur J Clin Invest 1990; 20:66-71). The authors merely mention that divalent cations must be present in the reaction mixture at a concentration of 2 mmol/l or 3 mmol/l (Mg.sup.2+) and do not describe a potential influence of ion concentration on the binding of endotoxin. The authors do not conclude that an increase in the ion concentration in the solution, and an indirectly related increase in apotransferrin-cation complexes, result in an improvement of endotoxin binding by apotransferrin. Thus these publications contain no indication whatever that a primary binding of cations to apotransferrin could be one of the preconditions for the binding of endotoxins to apotransferrin.
Endotoxin is a constituent of the cellular walls of gram-negative bacteria and is released only by the bacterial decay. It is a macromolecule with a molecular weight of up to 1.times.10.sup.6 Dalton, consisting mainly of sugar compounds and fatty acids. It may also include complexed protein residues from the wall of the bacteria. The endotoxin molecule consists of three structurally and immunobiologically different subregions:
Subregion 1, the O-specific chain, consists of several repetitive oligosaccharide units, each of which is made up of a maximum of 5 neutral sugars. The number of oligosaccharides present depends on the strain of bacteria; for example, the endotoxin of S. abortus equi used in our experiments has 8 oligosaccharides in this region.
Subregion 2, the core oligosaccharide, consists, among other things, of n-acetyl-glucosamine, glucose, galactose, heptose and 2-keto-3-desoxyoctone acid.
Subregion 3, The lipid A (MW 2,000 Dalton) consists of a phosphorylated D-glucosamine-disaccharide to which several--approx. 7-long-chain fatty acids are bound as amides and esters. The carrier of the toxic properties is the lipid A, whereby the toxic effects derive from several fatty acid residues in this region.
The size of the endotoxin molecule and its charge characteristics allow for complexing various compounds and proteins with the groups or side-chains of the three subregions in the endotoxin structure without this having any influence on its toxic properties. Normally, there is a protein bound to the lipid A, the so-called lipid A-associated protein. In most cases, separation of this protein component from the lipid A causes no change whatever in the toxic effect in some endotoxins. It was, however, found that binding of proteins onto many endotoxins can also result in a considerable increase in their toxicity (Rietschel E.TH. et al. (1982)): "Bacterial Endotoxins: Chemical Structure, Biological Activity and Role in Septicaemia", Scand. J. Infect. Dis. Suppl. 31: 8-21). For example, it was observed that certain endotoxins that have complexed a certain amount of protein can be 100 times as toxic as endotoxins from which the same protein was separated (Morrison DC, Oudes ZG, Betz J (1980): The role of lipid A and lipid A-associated protein in cell degranulation mechanisms. In: Eaker D. Wadstrom T (Eds). NATURAL TOXINS, pp 287-294, Pergamon Press. New York). In animal experiments as well, a decrease in toxic effect was observed when the protein was split off (Hitchcock PJ, Morrison DC. (1984). "The protein component of bacterial endotoxins". In: E.T. Rietschel (editor) HANDBOOK of ENDOTOXINS, Vol 1: Chemistry of Endotoxin, pp 339-375, Elsevier Science Publishers B.V.).
Free, i.e. biologically active, endotoxin cannot normally be detected in the blood of healthy subjects. In the following pathological conditions, however, increased amounts of biologically active endotoxin may occur in the blood:
a) Increased transfer of endotoxin from the intestinal tract into the blood on account of permeability disturbances in the intestinal wall, e.g. in severe enterocolitis, shock, or increased release in the course of enteral antibiotic therapy.
b) Reduced eliminetion of endotoxins by the liver, e.g. liver dysfunction partial liver resection.
c) Increased endotoxin release from a larger gram-negative focus such as could be found in treatment of peritonitis with antibiotics.
To protect organs from damage, plasma in healthy persons can inactivate the endotoxin that is continually transferred from the intestinal tract. An excessive endotoxin transfer into the blood brings about a rapid exhaustion of endotoxin-inactivating capacity of the plasma, so that even more biologically active endotoxin is found in the plasma, leading to the clinical signs of endotoxemia. If this condition continues, endotoxemia may lead to cell decay, and finally to organ failure. For these reasons, additional therapeutic measures are required to reduce blood endotoxin activity whenever increased endotoxin passage into the blood or reduced hepatic endotoxin elimination are to be expected.
To a certain extent, plasma endotoxin activity can be reduced by administering polyvalent 7S-IgG preparations, presumably due to the lipid A antibodies they contain. A further improvement in endotoxin neutralization is achieved by enriching the IgG preparation with immunoglobulin of the IgM fraction, in which a higher level of lipid A antibody titer is present (Appelmelk BJ et al., Microbiol Pathogenesis 1987; 2: 391-393).
Clinical studies (Baumgartner JD, et al. Prevention of gram-negative shock and death in surgical patients by antibody to endotoxin core glycolipid. Lancet 1985;ii: 59-63; Dunn DL, Priest BP, Condie RM. Protective capacity of polyclonal and monoclonal antibodies directed against endotoxin during experimental sepsis. Arch Surg 1988; 123:1389-1393; Ziegler EJ. et al. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. N Engl J Med 1991, 324: 429-436) have demonstrated that lethality of septicemia can be reduced by decreasing plasma endotoxin activity if monoclonal antibodies to the core or the lipid A portion of the endotoxin are administered.
An alternative to the neutralization of endotoxin by monoclonal antibodies is neutralization of the toxic effect of endotoxin by transferrin (DE 38 44 667). The combination of transferrin with polyvalent immunoglobulin preparations can achieve a synergistic effect as described in DE 38 42 143.
As described in E 38 44 667 in detail, the capacity of apotransferrin to neutralize endotoxin increases as the content of trivalent iron ions increases. In the organism, however, the iron can be split off from the transferrin by means of a reduction to Fe.sup.2+. Since the oxygen activation of enzyme-coupled reactions, i.e. formation of oxygen radicals by neutrophil granulocytes, can be stimulated by an increased presence of Fe.sup.2+ ions, it is possible that transferrin therapy may facilitate the undesirable formation of harmful oxygen radicals in the organism. However, non-enzymatic processes that lead to oxygen activation and the damaging oxygen radicals it entails, such as the so-called Haber-Weiss reaction (Haber F., Weiss J. Proc Royal Soc [A]1934; 147: 332-351) may be catalysed by the reduced divalent iron split off from transferrin (Carlin G., Djursater R. FEBS Lett 1984; 177: 27-30). The raised plasma protease activity in septicemia is another factor that can lead to a splitting of the transferrin molecule (Esparza I., Brock JH. Biochem Biophys Acta 1980; 622:297-304; Doring GM. et al. Infect Immun 1988; 56: 291- 293). The transferrin fragments that are released in this way and have complexed iron can also catalyze oxygen radical formation by neutrophil granulocytes (Bradley EB., Edeker BL. J Clin Invest 1991; 88: 1092-1102). The oxygen radicals can, on the one hand, cause direct damage to the cell membrane; on the other hand, they may damage the organism indirectly by increasing prostaglandin synthesis.